An ultra-rapid real-time RT-PCR method using the …...Address correspondence to: Kazuya Shirato, DVM, PhD. Senior Researcher Department of Virology III National Institute of Infectious

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Original Article

An ultra-rapid real-time RT-PCR method using the PCR1100 to detect

Severe Acute Respiratory Syndrome Coronavirus-2

1Kazuya Shirato, 1Naganori, Nao, 1Shutoku Matsuyama, 1Makoto Takeda and 2Tsutomu

Kageyama

1Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of

Virology III, and 2Influenza Virus Research Center, National Institute of Infectious

Disease, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan

Running title: Ultra-rapid real-time RT-PCR for SARS-CoV-2

Keywords: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ultra-rapid

real-time RT-PCR, PCR1100

Address correspondence to:

Kazuya Shirato, DVM, PhD.

Senior Researcher

Department of Virology III

National Institute of Infectious Diseases, Murayama Branch

4-7-1 Gakuen, Musashimurayama

Tokyo, 208-0011, Japan

E-mail: [email protected]

Tel: +81-42-561-0771

Fax: +81-42-567-5631

mailto:[email protected]

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著者一覧

白戸憲也〒208-0011 東京都武蔵村山市学園 4-7-1

国立感染症研究所ウイルス第 3 部

直亨則〒208-0011 東京都武蔵村山市学園 4-7-1

国立感染症研究所ウイルス第 3 部第 4 室

松山州徳〒208-0011 東京都武蔵村山市学園 4-7-1

国立感染症研究所ウイルス第 3 部第 4 室

竹田誠〒208-0011 東京都武蔵村山市学園 4-7-1


影山努〒208-0011 東京都武蔵村山市学園 4-7-1

国立感染症研究所インフルエンザウイルス研究センター第 2 室

責任著者連絡先

白戸憲也

〒208-0011 東京都武蔵村山市学園 4-7-1


Tel: 042-561-0771

Fax: 042-567-5631

E-mail: [email protected]

mailto:[email protected]

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Summary

The disease caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) in

Wuhan, China, in December 2019 is currently spreading rapidly worldwide. SARS-CoV-

2 is usually detected via real-time RT-PCR. However, as institutions/hospitals deal with

increasing numbers of specimens, a simpler detection system is required. Here, we present

an ultra-rapid, real-time RT-PCR assay for SARS-CoV-2 using the PCR1100 device.

Although this tests only one specimen at any one time, the amplification period is <20

min, with maintenance of the sensitivity and specificity of conventional real-time RT-

PCR performed using large instruments. The method will be very helpful if SARS-CoV-

2 testing is required a few times daily, for example to confirm virus-free status prior to

discharge.

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Introduction

The outbreak of coronavirus disease 2019 (COVID-19) caused by Severe Acute

Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) commenced in Wuhan, China, in

December 2019 and has rapidly spread worldwide (1, 2). As of June 1, 2020, 6,057,853

confirmed cases and 371,166 deaths have been reported globally (3). We previously

described genetic diagnostic methods for detection of SARS-CoV-2; these have been used

in Japanese prefectural and municipal public health institutes and quarantine depots (4).

As of June 1, 2020, 16,884 confirmed cases and 892 deaths have been reported in Japan

(3). The real-time RT-PCR assay takes several hours; specimens must be sorted and

numbered, RNA extracted, and RT-PCR performed. The data must be analyzed and

reported. This is impractical if testing is performed several times daily. As case numbers

increase, the total assay time must be shortened, both for new diagnoses and for

confirmation of virus-free status, which is required prior to patient discharge. Ultra-rapid,

real-time RT-PCR assays for Middle East respiratory syndrome coronavirus (MERS-

CoV) (5) and human orthopneumovirus (6) have been developed using the PCR1100, a

mobile real-time PCR device. Here, we used the device to develop an ultra-rapid, real-

time RT-PCR assay for SARS-CoV-2; we compared the assay to that of the conventional

real-time RT-PCR assay using the LightCycler.

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Materials and Methods

Viruses: The SARS-CoV-2 isolates (AI/I-004/2020, GISAID EPI_ISL_407084;

TY/WK-521/2020, EPI_ISL_408667; TY/WK-501/2020, EPI_ISL_408666) were used

(7). SARS coronavirus (the Frankfurt strain) was supplied by Dr. J. Ziebuhr, University

of Würzburg, Germany. The MERS-CoV EMC strain was kindly provided by Dr. Ron A.

M. Fouchier, Erasmus Medical Center, Rotterdam, The Netherlands. Human

orthopneumoviruses [respiratory syncytial virus (RSV), Long, A2, B WV/14617/85 (B1

wild type), and CH/18537] were obtained from the American Type Culture Collection

(ATCC; Manassas, VA, USA). Human coronavirus (HCoV)-229E isolates ATCC VR-740,

Sendai-H/1121/04, and Niigata/01/08 (8) were used. HCoV-NL63 (Amsterdam I) was

supplied by Dr. Lia van der Hoek, University of Amsterdam, the Netherlands. HCoV-

OC43 ATCC VR-1558 was used. Human respiroviruses [parainfluenza viruses (PIV) 1

(strain C35) and 3 (strain C243)] were obtained from ATCC. Adenoviruses (ADVs)

(serotype 3, strain G.B.; serotype 4, strain RI-67; and serotype 7, strain Gomen) were also

obtained from ATCC. Viruses were propagated and titrated using HEp-2, HeLa, RD, Vero,

VeroE6, LLC-Mk2, or Vero/TMPRSS2 cells (9); otherwise, copy numbers were

calculated by real-time RT-PCR (10). Influenza viruses [Flu; A/California/7/2009

(H1N1pdm), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008] were propagated

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and titrated using MDCK cells. Clinical isolates of HCoV-OC43 (Tokyo/SGH-36/2014,

LC315646; Tokyo/SGH-61/2014, LC315647; Tokyo/SGH-06/2015, LC315648;

Tokyo/SGH-65/2016, LC315649), HKU1 (Tokyo/SGH-15/2014, LC315650;

Tokyo/SGH-18/2016, LC315651), and NL63 (Tokyo/SGH-15/2017, LC488390;

Tokyo/SGH-24/2018, LC488388) were isolated and propagated using human bronchial

tracheal epithelial cells (Lifeline Cell Technology, Frederick, MD, USA), cultured, and

allowed to differentiate at an air-liquid interface, as previously described (11). Copy

numbers were calculated via virus-specific real-time RT-PCR (12). Clinical isolates of

RSV (A/NIID/2347/14, LC474556; A/NIID/2367/14, LC474557; A/NIID/2470/14,

LC474558; B/NIID/2472/14, LC474559; B/NIID/2474/14, LC474560), isolated using

HEp-2 cells, were also used (6). A clinical isolate of human metapneumovirus (HMPV;

IA10-2003] was obtained from ZeptoMetrix (https://www.zeptometrix.com/). Three

clinical isolates of previously reported hMPV (Sendai/0256/2015, Sendai/414/2013, and

Sendai/1052/2011) were used (13). Clinical isolates of PIVs (PIV1/ NIID/79081/1/2019,

PIV1/NIID/79082/2/2019, PIV2/ NIID/56606/2/2019, PIV2/ NIID/56607/1/2019,

PIV3/NIID/79133/1/2019), isolated using Vero/TMPRSS2 cells, were used.

Specimens and extraction of nucleic acids from viral stocks

https://www.zeptometrix.com/

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Pharyngeal swabs, nasal swabs, and sputum obtained during national diagnosis tests were

used. Nasopharyngeal and nasal swabs from Discovery Life Sciences (Los Osos, CA,

USA) were also used. These were used with the approval of the Research and Ethical

Committee for the Use of Human Subjects of the National Institute of Infectious Diseases,

Japan (approval #1001 and 1091). RNA was extracted from viral stocks using TRIzol LS,

TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the QIAamp Viral

RNA Mini Kit (Qiagen, Hilden, Germany) was used for extractions from specimens,

according to the manufacturer’s instructions. The M1 Sample Prep Cartridge kit for RNA

(Biomeme, Philadelphia, PA, USA) was also used according to the manufacturer’s

instructions. Viral DNA was extracted using the SimplePrep Reagent for DNA (Takara

Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. A positive control

RNA of ribonuclease (RNase)P-transcribed from a T7 promotor-incorporated PCR

template was also used in the validation tests.

Real-time RT-PCR

Primers and probes were designed with the aid of Primer 3 software (ver. 4.0,

http://bioinfo.ut.ee/primer3-0.4.0/) based on the sequence of a Japanese viral isolate

(Japan/TY/WK-521/2020, EPI_ISL_408667), as follows: SARS-CoV-2-NIID-N3-F1, 5′-

http://bioinfo.ut.ee/primer3-0.4.0/

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CATTGGCATGGAAGTCACAC-3′; SARS-CoV-2-NIID-N3-R5, 5′-

CAAAATGACTTGATCTTTGAAATT-3′; SARS-CoV-2-NIID-N3-P1, 5′- FAM-

TCGGGAACGTGGTTGACCTACACA-BHQ1-3′; SARS-CoV-2-NIID-S-F1, 5′-

CAGTCAGCACCTCATGGTGTA -3′; SARS-CoV-2-NIID-S-R1, 5′-

GGCAGGAGCAGTTGTGAAGT-3′; SARS-CoV-2-NIID-S-P1, and 5′- Cy5-

CATGTGACTTATGTCCCTGCACAAGAA-BHQ3-3′. The human RNase P gene served

as the internal control, by referring to a previous report from the US Centers for Diseases

Control (US-CDC) (14); forward primer, 5′-AGATTTGGACCTGCGAGCG-3′; reverse

primer 5′-GAGCGGCTGTCTCCACAAGT-3′; and probe HEX-5′-

TTCTGACCTGAAGGCTCTGCGCG-3′-BHQ. For ultra-rapid real-time RT-PCR, the

KAPA3G Plant PCR kit (Kapa Biosystems, Wilmington, MA, USA) and FastGene

Scriptase II (Nippon Genetics Co., Ltd. Tokyo, Japan) were used. The primer and probe

concentrations were as follows: NIID-N1-F1, 730 nM; NIID-N1-R5, 975 nM; NIID-N1-

P1, 695 nM; NIID-S-F1, 320 nM; NIID-S-R1, 410 nM; NIID-S-P1, 370 nM, RNaseP-F,

277.8 nM; RNaseP-R, 555.6 nM; and RNaseP-P, 416.7 nM. Primers and probes were

mixed in 0.8 L / reaction, in advance. The cycling conditions and reaction components

were as previously described (5) with a few modifications: 10 L of 2× buffer (Plant 3G

kit), 1.35 L of MgCl2 (Plant 3G kit), 1 L of FastGene Scriptase II, 1.5 L of DNA

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polymerase (Plant 3G kit), 0.2 L of dithiothreitol (100mM, FastGene Scriptase II), and

0.8 L of primer/probe mix. The following real-time RT-PCR conditions were set for the

PCR1100: 55°C for 180 sec; 95°C for 15 sec; and 50 cycles of 95°C for 5 sec and 61°C

for 7 sec. A Cq<48 value of <48 for the S set and <50 for the N3 set were considered

positive. When performing conventional real-time RT-PCR, we used both previously

reported sets [Sarbeco-N (15) and N2 (4)] and also the S set mentioned above.

Concentrations of the S set were as follows: NIID-S-F1, 400 nM; NIID-S-R1, 800 nM;

and NIID-S-P1, 200 nM. Conventional real-time RT-PCR was performed using the

QuantiTect Probe RT-PCR kit (Qiagen, Hilden, Germany) and a LightCycler 96 or 480

system (Roche, Basel, Switzerland) as previously described. Cp values <37.5 for the S

set and <40 for the Sarbeco-N and N2 sets were considered to be positive.

Results

First, assay sensitivities were validated using RNA from three Japanese isolates

(Table 1). The sensitivities of sets N2 and S were ~2 copies and that of the Sarbeco-N set

~10 copies in the conventional real-time RT-PCR assay; that of the S set was 5 copies and

that of the N1 set 15.8 copies on ultra-rapid real-time RT-PCR. Assay specificity was also

validated using other respiratory viruses; there was no cross-reaction with other

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respiratory viruses, including clinical isolates (Table 2).

The ultra-rapid, real-time RT-PCR assay was further validated using clinical

specimens, in comparison with the N2 and S sets analyzed on the LightCycler (Table 3).

Clinical specimens obtained during national infectious disease surveillance tests for

SARS-CoV-2 were used. Totals of 27 negatives (nos. 1–27) and 14 positives (nos. 32–

45) were confirmed using the national tests (4). The ultra-rapid test yielded identical

results, detecting SARS-CoV-2 RNA in either or both of the N1 and S sets of positive

specimens, and not in the negative specimens. For the evaluation of cross-reaction in

clinical specimens diagnosed as other respiratory pathogens, pre-diagnosed specimens

were obtained from Discovery Life Sciences (Los Osos, CA, USA). The specimens were

collected as part of routine laboratory analysis in various countries and pathogens were

diagnosed by various systems, such as virus isolation, BinaxNow kits, Dimension Vista

system, and GenMark’s eSensor Respiratory Viral Panel (No. 46-61). Four undiagnosed

specimens from Discovery Life Sciences were also used (No. 28-31). No cross-reactivity

with specimens from patients diagnosed with other respiratory viruses was evident.

When detecting RNA viruses, RNA extraction is time-consuming. The M1 Sample

Prep Cartridge kit for RNA (Biomeme, Philadelphia, PA, USA) has been used to detect

human orthopneumovirus (6); RNA extraction required only 5 min using the pumping

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action of a filter-attached syringe. We used this method to extract RNA from 400 L

amounts of SARS-CoV-2 specimens. In all, 18 negatives and 13 positives, confirmed

using the N2 set to assay RNAs purified via a Qiagen spin column, were used. All

negative specimens remained negative in both the ultra-rapid and conventional real-time

RT-PCR assays. However, nine of 13 were positive by the conventional real-time RT-PCR

assay and five of 13 positive by the ultra-rapid real-time RT-PCR assay, suggesting that

the M1 Sample Prep Cartridge kit reduced SARS-CoV-2 detection rate.

Discussion

This study showed that SARS-CoV-2 can be detected using the ultra-rapid real-time RT-

PCR assay in <20 min with a sensitivity and specificity similar to those of conventional

real-time RT-PCR. The ultra-rapid assay is also very practical. Although the device tests

only one specimen at a time, this is an advantage when viral testing is required for only a

few or several cases a few times a day, for example to confirm virus-free status prior to

discharge. The PCR1100 is inexpensive compared to a real-time RT-PCR device (about

800,000 JPY); therefore, several devices could be used to replace a very expensive real-

time RT-PCR platform.

RNA extraction is time-consuming event, therefore, The M1 Sample Prep Cartridge

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kit for RNA (Biomeme) was used like detection of human orthopneumovirus (6).

However, the result of this study showed lower detection rate was seen in M1 Sample

Prep kit as compared to QIAamp viral RNA Mini kit. This was not the case for human

orthopneumovirus detection (6). It is possible that the SARS-CoV-2 specimens had fewer

copies of viral RNA than the orthopneumovirus specimens. Indeed, the false-negatives

exhibited high Cp values, averaging 33.5 and 32.5 for conventional and ultra-rapid real-

time RT-PCR, respectively. We previously isolated virus from VeroE6/TMPRSS2 cells;

the Cp was <32 (7), suggesting that use of the M1 Sample Prep Cartridge kit afforded a

SARS-CoV-2 detection sensitivity equal to that of the VeroE6/TMPRSS2 cell culture

approach (8). Alternative methods must be developed for the improvement of difficulty

of RNA extraction step.

Competing Interests

No author has any competing interest.

Acknowledgment

We thank all staff of NCGM and local prefectural laboratories for specimen

collection/transportation/assay, and their strong support. We also thank all staff from NIID

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and FETP for their invaluable support. We acknowledge all staff who cared for

patients/returnees. We also thank all staff from MHLW and the quarantine sectors for help

with administration and our investigations.

Funding

This work was supported by Grants-in-Aid (nos. 19fk0108110j0001 and

20fk0108117j0101) from the Japan Agency for Medical Research and Development.

References

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disease in China. Nature. 2020;579:265-9.

2. Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new

coronavirus of probable bat origin. Nature. 2020;579:270-3.

3. WHO. Novel Coronavirus (2019-nCoV) situation reports. Available at:

<https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/>.

Accessed June 2, 2020.

4. Shirato K, Nao N, Katano H, et al. Development of Genetic Diagnostic Methods for

Novel Coronavirus 2019 (nCoV-2019) in Japan. Jpn J Infect Dis. 2020; doi:

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10.7883/yoken.JJID.2020.061.

5. Shirato K, Nao N, Matsuyama S, et al. Ultra-Rapid Real-Time RT-PCR Method for

Detecting Middle East Respiratory Syndrome Coronavirus Using a Mobile PCR Device,

PCR1100. Jpn J Infect Dis. 2020;73:181-6.

6. Shirato K, Nao N, Kawase M, et al. An ultra-rapid real-time RT-PCR method for

detecting human orthopneumovirus using PCR1100. Jpn J Infect Dis. 2020; doi:

10.7883/yoken.JJID.2020.182.

7. Matsuyama S, Nao N, Shirato K, et al. Enhanced isolation of SARS-CoV-2 by

TMPRSS2-expressing cells. Proc Natl Acad Sci U S A. 2020;117:7001-3.

8. Shirato K, Kawase M, Watanabe O, et al. Differences in neutralizing antigenicity

between laboratory and clinical isolates of HCoV-229E isolated in Japan in 2004-2008

depend on the S1 region sequence of the spike protein. J Gen Virol. 2012;93:1908-17.

9. Shirogane Y, Takeda M, Iwasaki M, et al. Efficient multiplication of human

metapneumovirus in Vero cells expressing the transmembrane serine protease TMPRSS2.

J Virol. 2008;82:8942-6.

10. Kaida A, Kubo H, Takakura K, et al. Associations between co-detected respiratory

viruses in children with acute respiratory infections. Jpn J Infect Dis. 2014;67:469-75.

11. Shirato K, Kawase M, Matsuyama S. Wild-type human coronaviruses prefer cell-

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surface TMPRSS2 to endosomal cathepsins for cell entry. Virology. 2018;517:9-15.

12. Owusu M, Annan A, Corman VM, et al. Human coronaviruses associated with upper

respiratory tract infections in three rural areas of Ghana. PLoS One. 2014;9:e99782.

13. Nao N, Sato K, Yamagishi J, et al. Consensus and variations in cell line specificity

among human metapneumovirus strains. PLoS One. 2019;14:e0215822.

14. Emery SL, Erdman DD, Bowen MD, et al. Real-time reverse transcription-

polymerase chain reaction assay for SARS-associated coronavirus. Emerg Infect Dis.

2004;10:311-6.

15. Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-

nCoV) by real-time RT-PCR. Euro Surveill. 2020;25:2000045.

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Table 1. Sensitivity of ultra-rapid real-time RT-PCR for SARS-CoV-2.

Set Isolate names Positive/tested Sensitivity (copies)

Single assay

(LightCycler)

N2

copies 2500 250 25 2.5 NC**

2.5

AI/I-004/2020 4/4 4/4 4/4 0/4

0/6 TY/WK-521/2020 4/4 4/4 4/4 3/4

TY/WK-501/2020 4/4 4/4 4/4 3/4

Total 12/12 12/12 12/12 6/12 0/6

S

AI/I-004/2020 3/3 3/3 3/3 1/3

0/15

2.2

TY/WK-521/2020 3/3 3/3 3/3 1/3

TY/WK-501/2020 3/3 3/3 3/3 3/3

Total 9/9 9/9 9/9 5/9 0/15

Sarbeco-

N

AI/I-004/2020 3/3 3/3 2/3 0/3

0/5

10.2

TY/WK-521/2020 3/3 3/3 2/3 0/3

TY/WK-501/2020 3/3 3/3 3/3 1/3

Total 9/9 9/9 7/9 1/9 0/5

Multi assay

(PCR1100)

N3

copies 5000 500 50 5 NC

15.8

AI/I-004/2020 2/2 2/2 1/2 1/2

0/5 TY/WK-521/2020 2/2 2/2 2/2 0/2

TY/WK-501/2020 2/2 2/2 2/2 0/2

Total 6/6 6/6 5/6 1/6 0/5

S

AI/I-004/2020 2/2 2/2 2/2 1/2

0/5 5

TY/WK-521/2020 2/2 2/2 2/2 1/2

TY/WK-501/2020 2/2 2/2 2/2 1/2

Total 6/6 6/6 6/6 3/6 0/5

RNase P 6/6 6/6 6/6 6/6 6/6

*: Each sample contained 500 copies of control RNA (encoding RNase P).

**: Negative control (NC) is water contains 10g/ml of Yeast RNA as carrier.

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Table 2. Specificity of ultra-rapid real-time RT-PCR for SARS-CoV-2.

Single assay Multi assay

Virus Name of isolate Amount/reaction S set N1 set S set RNase P*

Coronaviruses

SARS-CoV-2 Japan/TY/WK-521/2020 2.5×105 copies + + + +

SARS-CoV Frankfurt-1 5×104 copies - - - +

MERS-CoV EMC 2.5×105 copies - - - +

Human coronaviruses (HCoV)

HCoV-229E VR-740 1.8×109 copies - - - +

Sendai-H/1121/04 4.9×106 copies - - - +

Niigata/01/08 2×105 copies - - - +

HCoV-NL63 Amsterdam I 2×109 copies - - - +

Tokyo/SGH-15/2017 2.7×106 copies - - - +

Tokyo/SGH-24/2018 4.8×105 copies - - - +

HCoV-OC43 VR-1558 5.1×1010 copies - - - +

Tokyo/SGH-36/2014 6.8×107 copies - - - +

Tokyo/SGH-61/2014 3.3×108 copies - - - +

Tokyo/SGH-06/2015 1.1×108 copies - - - +

Tokyo/SGH-65/2016 2.9×108 copies - - - +

HCoV-HKU1 Tokyo/SGH-15/2014 1.5×108 copies - - - +

Tokyo/SGH-18/2016 3.3×107 copies - - - +

Influenza viruses

H1N1pdm A/California/7/2009 1×105 copies - - - +

H3N2 A/Victoria/210/2009 8.4×104 copies - - - +

B B/Brisbane/60/2008 4.2×105 copies - - - +

Paramyxoviruses

Human respirovirus 1 (PIV1) C-35 4 ×105 copies - - - +

NIID/79081/1/2019 1.3 ×103 coipes - - - +

NIID/79082/2/2019 2.4 ×104 copies - - - +

Human respirovirus 3 (PIV3) C-243 5.1 ×107 copies - - - +

NIID/79133/1/2019 3.6 ×103 coipes - - - +

Human Rubulavirus 2 (PIV2) NIID/56606/2/2019 7.3×102 TCID50 - - - +

NIID/56607/1/2019 7.3×101 TCID50 - - - +

Pneumoviruses

Human orthopneumovirus Long 5.7×104 copies - - - +

(Respiratory syncytial virus, RSV) A2 2.5 ×106 copies - - - +

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CH/18537 4.8×105 copies - - - +

B1 2.5 ×106 copies - - - +

A/NIID/2347/14 5 ×104 copies - - - +

A/NIID/2367/14 5 ×104 copies - - - +

A/NIID/2470/14 5 ×104 copies - - - +

B/NIID/2472/14 5 ×104 copies - - - +

B/NIID/2474/14 5 ×104 copies - - - +

Human metapneumovirus IA10-2003 7.5 ×106 IU - - - +

(hMPV) Sendai/0256/2015 2 ×105 IU - - - +

Sendai/414/2013 3 ×106 FFU - - - +

Sendai/1052/2011 3 ×106 IU - - - +

Adenoviruses (ADV)

ADV 3 G.B. 7×105 TCID50 - - - +

ADV 4 RI-67 7×105 TCID50 - - - +

ADV 7 Gomen 7×105 TCID50 - - - +

*: Each sample contained 500 copies of control RNA (encoding RNase P).

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Table 3. SARS-CoV-2 detection using clinical specimens.

Single assay

(Cp value)

Multi assay

(Cq Value)

No. Diagnosed pathogen Specimen type N2 set S set N1 set S set RNase P

1 None Pharyngeal swab - - - - 38
























25 None Nasal swab - - - - 37

26 None Sputum - - - - 32

27 None Sputum - - - - 35

28 Undiagnosed Nasopharyngeal swab - - - - 36




32 SARS-CoV-2 Pharyngeal swab 21.8 22.4 28 28 34

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41 SARS-CoV-2 Pharyngeal swab 37.4 36.2 - 45 38




45 SARS-CoV-2 Sputum 22.5 23.5 31 32 33

46 ADV C Nasopharyngeal swab - - - - 37

47 ADV C Nasopharyngeal swab - - - - 39

48 hMPV Nasopharyngeal swab - - - - 37

49 hMPV Nasopharyngeal swab - - - - 34

50 Influenza A Nasopharyngeal swab - - - - 37

51 Influenza A Nasopharyngeal swab - - - - 37

52 PIV1 Nasopharyngeal swab - - - - 35






58 RSV A Nasopharyngeal swab - - - - 32

59 RSV B Nasopharyngeal swab - - - - 31

60 RSV A Nasal swab - - - - 31

61 RSV B Nasal swab - - - - 31

Documents

An ultra-rapid real-time RT-PCR method using the …...Address correspondence to: Kazuya Shirato, DVM, PhD. Senior Researcher Department of Virology III National Institute of Infectious